Searches for the nutrition requirements of three strains of Brevibacterium ammoniagenes reported in the previous paper were carried out with an aim of achieving the striking accumulation of L-glutamic acid and the large multipication of cells. It was recognized that all three strains required both biotin and thiamine, together with amino acids such as histidine or cysteine, for their good growth and extracellular L-glutamic acid accumulation. The quantity of biotin required for remarkable growth of these microorganisms was quite different from that for the maximum production of L-glutamic acid. This result, however, did not apply in the case of thiamine. It was also confirmed that, of 18 amino acids, histidine and cysteine were the msot effective organic nitrogen sources, while the most available inorganic ammonium salt resulting in a large amount of L-glutamic acid-production and considerable cell gorwth was found to be only urea. Maximum accumulation of extracellular L-glutamic acid, more than 50%(w/w) of the initial sugar content, could be obtained from fermentation in the medium containing wheat-bran extract(Brev. ammoniagenes T-1 and Brev.ammoniagenes Y-2) or rice-bran extract(Brev. ammoniagenes YR-2), which confirmed us a possibility that these bacteria might be employed for industrial fermentation of L-glutamic acid.

It is a well-known fact that an isolation of non-sporeforming anaerobes, considered normal flora in man ordinarily but causes serious infections sometimes, is a dificult procedure because of their great oxygen sensitivity. Among the many techniques employed in clinical laboratories, despite of its high expenses, the GasPak method has been most widely used because of its relative simplicity. On the other hand, the steel wool method has gained a good reputation recently. This technique makes it possible to treat individual plate so that any single specimen can be promptly cultured anaerobically. The procedure is quite simple and the expenses are negligible. In the present study it is to compare these two methods as to their efficiency of anaerobic cultivation using 13 VPI strains of non-sporeforming amaerobic bacteria. Among the 13 species the following 11, Bacteroides fragilis ss. fragilis, B. fragilis ss. thetaiotaomicron, propionibacterium acnes, Eubacterium limosum, E. lentum, peptococcus asaccharolyticus, Pc. prevotii, Pc. magnus, peptostreptococcus anaerobius, Ps. intermedius nad Veillonella parvula, grew well with the steel wool method whose colony numbers reaching 57 to 119% of those with GasPak method. The remaining two species, Fusobacterium nucleatum and F.necrophorum, did not grow well with the steel wool method showing the colony numbers were only 0.4% of those with GasPak method in the case of Fusobacterium nucleatum. In the case of Fusobacterium necrophorum, very few colonies developed even with a heavy inoculation. As to the size of colonies, there were no significant difference between these two methods.

Taxonomic relations among three species of some edible fleshy fungi(Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes) in the family Tricholomataceae were examined by using polyacrylamied gel disc electrophoresis. The soluble, crude extract of mycelium grown on potato sucrose broth was subjected to electrophoresis. Similarities in the protein bands for each isolated of one species were compared with those for others. In the banding patterns there was a closer relationship between isolates within one species than among isolates of different species. However, the isozyme patterns obtained from each isolate of Peurotus ostreatus (esterase, peroxidase, tyrosinase) were appeared to represent the degree of geographical variability within one species.

A small amount of Duco cement or Elmer's clear cement was dropped on a slide glass and immediately spread with a glass rod or woden stick a thin film on the surface. After approximately 1 minute a small amount of rubber cement was spread on the top of the film of Duco cement using the same method as described earlier. It was important that the rubber cement be smeared before the Duco cement dried out. These two kinds of cements must not be mixed. It was better to make the film of the rubber cement slightly thicker than the film of Duco cement. This composite film may be used up to several months after preparation. The sample leaf was placted on the slide, prepared with adhesive and the leaf surface was passed on the film with the thumb. The pressing was done so the leaf surface was completely in contact with the film. Then the leaf was peeled from the slide.